Agarose is the principal constituent of agar. Agarose is a polysaccharide that has a structure in which D-galactose and 3,6-anhydro-L-galactose are alternately linked together through α-1,3 bonds and β-1,4 bonds. One must degrade agarose into smaller molecules in order to produce oligosaccharides from agar. For this purpose, methods in which agarose is chemically degraded and methods in which agarose is enzymatically digested are conventionally known. In a chemical degradation method, agarose can be hydrolyzed using an acid. In this case, α-1,3 bonds are mainly cleaved. Two types of enzymes, β-agarases which cleave β-1,4 bonds in agarose and α-agarases which cleave α-1,3 bonds in agarose, are known to digest agarose.
Oligosaccharides obtained by cleaving agarose at β-1,4 bonds are called neoagarooligosaccharides. Neoagarooligosaccharides have D-galactose at their reducing ends and their degrees of polymerization are expressed by even numbers. On the other hand, oligosaccharides obtained by cleaving agarose at α-1,3 bonds are called agarooligosaccharides. Agarooligosaccharides have 3,6-anhydro-L-galactose at their reducing ends and their degrees of polymerization are expressed by even numbers. Recently, it was shown that agarooligosaccharides which have 3,6-anhydro-L-galactose at their reducing ends have physiological activities such as an apoptosis-inducing activity, a carcinostatic activity, various antioxidant activities, an immunoregulatory activity, an antiallergic activity, an anti-inflammatory activity and an activity of inhibiting α-glycosidase (WO 99/24447, Japanese Patent Application No. 11-11646). Based on the physiological activities, pharmaceutical compositions and functional foods or drinks containing the agarooligosaccharides as their active ingredients can be provided.
It is difficult to control the sizes of produced oligosaccharides in a method in which agarose is chemically degraded. In particular, it is quite difficult to selectively produce smaller oligosaccharides with low degrees of polymerization (e.g., T. Tokunaga et al., Bioscience & Industry, 49:734 (1991)). If a β-agarase is used, only neoagarooligosaccharides which do not have the above-mentioned physiological activities can be obtained because this enzyme cleaves only β-1,4 bonds.
It is expected that agarooligosaccharides having physiological activities are produced by using an α-agarase which has an activity of cleaving α-1,3 bonds. Known α-agarases include enzymes produced by a marine Gram-negative bacterial strain GJ1B (Carbohydrate Research, 66:207-212 (1978); this strain is indicated as Alteromonas agarlyticus GJ1B in European Journal of Biochemistry, 214:599-607 (1993)) and a bacterium of genus Vibrio (JP-A 7-322878; strain JT0107-L4). However, it is impossible to produce agarobiose which has notable physiological activities by using the α-agarase derived from Alteromonas agarlyticus GJ1B because the enzyme cannot digest hexasaccharides or shorter oligosaccharides. Furthermore, the α-agarase derived from the bacterium of genus Vibrio cannot be used for the production of agarooligosaccharides using agarose as a raw material because this enzyme exhibits its activity only on hexasaccharides and shorter oligosaccharides and does not act on agarose at all.
The present inventors studied intensively in order to obtain an enzyme that cleaves α-1,3 bonds in agarose and generates agarooligosaccharides having notable physiological activities and found two microorganisms that produce enzymes having properties suitable for this purpose. The enzymes produced by these microorganisms were isolated and their physical and chemical as well as enzymatic properties were shown. Furthermore, the present inventors isolated genes for the two enzymes, and found a method for conveniently producing polypeptides having α-agarase activities by means of genetic engineering using the genes (WO 00/50578). If one intends to obtain agarooligosaccharides by treating dissolved agarose with one of the two α-agarases, the reaction temperature needs to be lowered to about 40° C. because the optimal reaction temperatures of the enzymes are about 40° C. In this case, if the agarose concentration is high, agarose may be solidified and the enzymatic reaction may be prevented. Therefore, if such an enzyme is to be used, the treatment needs to be carried out using agarose at a low concentration. Accordingly, agarose at a high concentration cannot be treated and the productivities of agarooligosaccharides are low. Thus, a thermostable α-agarase that has an activity at a high temperature at which agarose is not solidified even if agarose is dissolved at a high concentration has been desired.
On the other hand, extraction of a nucleic acid or the like which has been subjected to treatment with a restriction enzyme or an amplification reaction from an agarose gel after agarose gel electrophoresis is widely carried out in a field of genetic engineering. A β-agarase having an optimal temperature of about 37° C. has been conventionally used for the procedure. Also in this case, the reaction temperature needs to be lowered depending on the optimal temperature of the enzyme, and the agarose concentration has to be lowered by dissolving an agarose gel containing a nucleic acid or the like to be treated in a large volume of water in order to prevent agarose from solidifying. In this case, the concentration of the nucleic acid or the like to be recovered is also lowered inevitably. The lowered concentration causes problems because it results in lowered recovery and lowered efficiency of agarose digestion as well as prolonged procedure. An agarase that can be used for a reaction at a temperature higher than the conventional one is necessary in order to solve the problems. A β-agarase derived from Flavobacterium sp. strain NR19 as described in U.S. Pat. No. 5,869,310 may be known as such an agarase, although its optimal temperature is not so high and the thermostability is insufficient. Thus, a thermostable β-agarase which has an activity at a high temperature at which agarose at a high concentration is not solidified has been desired.
As described above, prior art has problems regarding production of agarooligosaccharides and extraction of a material such as a nucleic acid from an agarose gel.